Progress in understanding the intricate development of the human nervous system and elucidating the mechanisms of mental disorders in patients has been greatly limited by restricted access to functional human brain tissue. While studies in rodents have provided important insights into the fundamental principles of neural development, we know little about the cellular processes responsible for the massive expansion of the cerebral cortex in primates, nor many of its human specific features. In recent years, a paradigm shift has been achieved in the field with the introduction of cellular reprogramming—a process during which terminally differentiated somatic cells can be converted into pluripotent stem cells, named human induced pluripotent stem cells (hiPSC). These hiPSCs can be generated from any individual and, importantly, can be directed to differentiate in vitro into all germ layer derivatives, including neural cells.
While the methods and efficiency of generating hiPSCs have been significantly improved and standardized across laboratories, the methods for deriving specific neuronal cell types and glia remain challenging. Over the past decade, improvements in neural specification and differentiation protocols of pluripotent stem cells in monolayer have led to the generation of a variety of cell types. Nonetheless, two-dimensional (2D) methods are unlikely to recapitulate the cytoarchitecture of the developing three-dimensional (3D) nervous system or the complexity and functionality of in vivo neural networks and circuits. Moreover, these methods are laborious and costly, have limited efficiency and give rise to relatively immature neurons.
Rodent and human in vitro corticogenesis often appears incomplete and because synaptogenesis requires the presence of glial cells, studying synaptic function in hiPSC-derived neurons requires co-culture with astrocytes. This is currently achieved by separately differentiating neurons and glia and subsequently co-culturing them, or by plating hiPSC-derived neurons on a monolayer of rodent astrocytes. These needs have spawned 3D approaches for generating organoid cultures containing mixed ectodermal derivatives. Although these methods recapitulate many aspects of corticogenesis and display a level of self-organization beyond what is possible in 2D cultures, there are several limitations including the need for: (1) controlled specification and generation of neural cell types; (2) cortical lamination and the generation of equal proportions of superficial and deep layer neurons; (3) generation of non-reactive astrocytes; (4) robust synaptogenesis and spontaneous synaptic activity; (5) organization of a functional neural network that can be perturbed and probed using intact preparations; and (6) reproducibility between hiPSC lines/clones and within and across differentiations.
Pharmaceutical drug discovery, a multi-billion dollar industry, involves the identification and validation of therapeutic targets, as well as the identification and optimization of lead compounds. The explosion in numbers of potential new targets and chemical entities resulting from genomics and combinatorial chemistry approaches over the past few years has placed enormous pressure on screening programs. The rewards for identification of a useful drug are enormous, but the percentages of hits from any screening program are generally very low. Desirable compound screening methods solve this problem by both allowing for a high throughput so that many individual compounds can be tested; and by providing biologically relevant information so that there is a good correlation between the information generated by the screening assay and the pharmaceutical effectiveness of the compound.
Some of the more important features for pharmaceutical effectiveness are specificity for the targeted cell or disease, a lack of toxicity at relevant dosages, and specific activity of the compound against its molecular target. The present invention addresses this issue.
Publications.
Methods to reprogram primate differentiated somatic cells to a pluripotent state include differentiated somatic cell nuclear transfer, differentiated somatic cell fusion with pluripotent stem cells and direct reprogramming to produce induced pluripotent stem cells (iPS cells) (Takahashi K, et al. (2007) Cell 131:861-872; Park I H, et al. (2008) Nature 451:141-146; Yu J, et al. (2007) Science 318:1917-1920; Kim D, et al. (2009) Cell Stem Cell 4:472-476; Soldner F, et al. (2009) Cell. 136:964-977; Huangfu D, et al. (2008) Nature Biotechnology 26:1269-1275; Li W, et al. (2009) Cell Stem Cell 4:16-19).
Foot-print free derivation of astrocytes is described by Mormone et al. (2014) Stem Cells and Development. Induction of neural cells from pluripotent cells is described by Yuan et al. (2013) Stem Cell Research and Therapy 4:73.